Electrophoresis involves the separation of charged molecules in an electric field. It is based upon the principle that an electric field will cause charged molecules to migrate into separate factions. Usually, separation of the charged molecules is based on the strength of the electrical field and the net charge, size and shape of the molecules. The rate of separation can also be based on other parameters, such as the isoelectric points, ionic strength, viscosity, and temperature of the medium in which the charged molecules are moving. Since proteins and other biological molecules, such as DNA, RNA, enzymes, carbohydrates and the like are charged, electrophoresis techniques are ideal to separate them for either analytical or preparative purposes.
Electrophoresis is generally performed in gels cast in tubes, slabs or on flat beds. A tube gel unit is formed in a glass tube which is typically 12 cm. in length and between about 3 and 5 mm. in internal diameter. In a gel slab, the gel is formed between two non-conducting plates spaced apart by two spacer strips at the edges and clamped together to make a water-tight seal. The gel tube units and gel slab cassettes are mounted vertically. The flat bed gel units, on the other hand, are mounted horizontally since the gels are poured on horizontal surfaces and they do not include top plates.
Gel electrophoresis devices may be broadly categorized as vertical gel electrophoresis devices or as horizontal gel electrophoresis devices. Typically, the vertical gel electrophoresis devices include a bottom tank and a top tank removably situated inside the bottom tank so as to provide two spaces for electrolytes, i.e., a first or lower space between the respective walls of the bottom and top tanks and a second or upper space inside the top tank. Both tanks include a vertically extending electrophoresis gel slab cassette, which contains a gel slab for separation of a mixture of charged biological molecules, and separate electrodes. In the electrophoresis devices, the only intended path for electricity is from the electrode in the top space to an electrode in the bottom space, via electrolytes in the top space, the gel and electrolytes in the bottom space, in that order. Buffer solutions are generally selected to function as the electrolytes in the tanks. To prevent the gel electrophoresis devices from shorting out and to force the electric current through the gel slab, it is imperative that the buffer solution in the top space remain separated at all times from the buffer solution in the bottom space. In other words, the electrical connection between the two electrodes is only through the gel slab.
As indicated, electrophoresis cassettes typically include a slab of gel cast in a sandwich-type arrangement between two die-electric or non-conducting flat plates, such as glass, to form a sheet of gel between the glass surfaces. Clear glass plates are generally selected to permit monitoring of the gel-forming solution as it is injected into the space between the plates, as well as monitoring of the finished gel as electrophoresis is taking place. While the gels used in the different types of electrophoresis cassettes may vary in shape, a common gel configuration is that of a thin, flat slab being generally of uniform thickness. The glass plates of electrophoresis cassettes are typically separated by thin, flat, rectangular-shaped spacer strips positioned between the glass plates and along their opposing vertical side edges. The electrophoresis cassettes are generally held together by clamps at each vertical edge to form the water-tight seals. To accomplish this, the clamps typically extend along the entire length of the opposing vertical side edges of the glass plates of the electrophoresis cassettes.
In performing an electrophoresis separation of a mixture of charged biological molecules, the surfaces at each end of the gel slab are connected to separate electrodes via the buffer solutions. A potential is applied across the gel slab by connecting each buffer solution to opposite polarities of a voltage source. The mixture of charged biological molecules to be sorted is placed at the negative electrode end of the gel slab, usually in preformed wells. The electrical field applied across the gel slab reacts with the negative charges on the biological molecules to provide a force propelling the charged biological molecules through the gel slab towards the positive electrode. Smaller charged biological molecules have less resistance to travelling through the gel slab than larger charged biological molecules, resulting in a separation and sorting of the biological molecules by size as they migrate through the gel slab.
Following electrophoresis runs, the gels can be analyzed by staining or autoradiography followed by densitometry, or by blotting to a membrane for nucleic acid hybridization, autoradiography or immunodetection. The autogradography method relied upon to view the electrophoretic separation of charged biological molecules in a gel slab involves, for instance, the use of radio-labeled molecules. Typically, the gel slab used for electrophoresis is removed from its cassette following the electrophoresis run and placed along side a photographic medium which is exposed by the radioactive emissions of the radio-labeled biological molecules. Developing the emissions in the photographic medium produces a series of stripes representative of the position of each set of radio-labeled biological molecules. In other words, the migration patterns of multiple rows obtained after the migration, i.e., a group of zones formed by electrophoresis on the gel slab, is recorded as an autoradiogram. When the mixture of charged biological molecules consists of DNA or RNA molecules, the base arrangement of the DNA or RNA molecules are determined by comparing positions of the separated zones in the respective rows with one another.
In the more common method relied upon to view the electrophoretic separation of charged biological molecules in a gel slab, it involves the use of a stain to stain the migrated molecules. This method generally involves removing the gel slab used for electrophoresis from its cassette following the electrophoresis run and exposing it to, for example, Coomassie Blue to stain the separated fractions to also produce a series of visible stripes representative of the position of each set of stained biological molecules. The molecular weights of the charged biological molecules, such as proteins, can be determined by comparing positions of the stained stripes in the respective rows with one another and against a row of visible stripes formed with markers having known molecular weights. In addition to Coomassie Blue, the separated molecules may be stained by photographic amplification systems using silver or other first row transition metals.
The comparison in either of the above recited methods is carried out based o the electrophoresis principle that charged biological molecules having equal molecular weights, charges and shapes migrate by equal distances if the electrophoresis is started from the same line and under the same conditions, such as ionic strength, viscosity and temperature.
While vertical electrophoresis gel slab cassettes have been successfully used up to now to carry out electrophoresis, they are not without drawback. During the initial assembly, the vertical electrophoresis gel slab cassettes available heretofore are generally assembled with tape running along the entire opposing vertical side edges of the two glass plates to hold the cassettes together. This obviously is a labor intensive and inconvenient method to assemble the cassettes. In addition, certain tapes available are not suitable to use due to the incompatibility between the adhesives on the tapes and the buffer solutions selected to carry out the electrophoresis. More importantly, when it is time to remove the gel slabs from the cassettes following the electrophoresis runs, the tape must be cut with, for example, a razor blade or sharp knife, since it will often tear, if peeled uncut, making it more difficult to remove. Another disadvantage associated with this method of opening the cassettes is the risk that the technicians handling the cassettes will cut themselves. The significance of this risk is underscored by the fact that the electrophoresed biological materials are often body fluids which may be contaminated with infectious diseases, such as AIDS, hepatitis and herpes.
In one attempt to overcome this problem, U.S. Pat. No. 4,929,329 provides a cassette with a strip, having top and bottom tabs and being positioned on the face of the top glass plate for cooperation with a monofilament, loop-shaped spacer positioned between the two glass plates for facilitating the replacement of the monofilament spacers when reassembling and filling new cassettes. Unfortunately, the cassettes disclosed in U.S. Pat. No. 4,929,329 cannot be conveniently stacked atop one another when such a strip is positioned on the front faces of the top glass plates. In addition, the spacers selected are monofilaments in unconventional loop-shaped form, so that the strips can be connected to the spacers.
Consequently, there is a demand in the electrophoresis industry for electrophoresis gel slab cassettes which can be easily and conveniently assembled and opened following electrophoresis without having to resort to razor blades or knives, so that the gel slabs can be removed therefrom without infectious risk and processed for preparing and/or analyzing the biological molecules electrophoresed therein.